Arsenic (As) is an inorganic environmental contaminant of major concern due to its ubiquitous presence. Chronic exposure to arsenic frequently results in peripheral vascular disease, as well as skin, lung, bladder, and kidney cancer. Recent evidence demonstrates that arsenite inhibits the production of plasmin which is necessary to dissolve blood clots in the vasculature. Our published studies with arsenic have demonstrated that the serine/threonine kinase, MEKK4, is involved in arsenic signal transduction, along with the calcium binding protein, annexin II. To understand the molecular mechanism by which arsenic causes its deleterious effects on the vascular system and how the activity of annexin II contributes to arsenic toxicity, our efforts have focused on characterizing the proteins that interact with and are regulated by annexin II. The hypothesis of this proposal is that annexin II plays a role in promoting arsenic-dependent peripheral vascular disease by modulating the formation of plasmin. To test this hypothesis, we propose the following specific aims: 1. To characterize arsenic-dependent post-translational modifications of annexin II. We propose that arsenic methylation produces homocysteine, which chemically modifies annexin II, thus inhibiting the production of plasmin and promoting clotting of the microvasculature. We propose that homocysteine-annexin II may be an indicator of arsenic exposure and we will explore the possibility of using homocysteine-annexin II as a biomarker. 2. To characterize the interaction between annexin II and MEKK4. Our data indicate interaction between annexin II and MEKK4, resulting in MEKK4 activity. Since MEKK4 appears to function downstream of annexin II, the biochemical association between these proteins will be mapped to specific domains. 3. To characterize arsenic-dependent regulation of PAI-1 and PAI-2. We propose that the ERK MAP kinase signaling pathway, regulated by annexin II and MEKK4, may be a mechanism for arsenic-dependent regulation of PAI expression. 4. To assess specific functions of annexin II. We will create a tissue-specific knockout mouse, selectively disrupting annexin II expression in smooth muscle cells, to evaluate arsenic toxicity in a whole animal. The proposed research will provide a further understanding of the importance of the arsenic-induced signaling processes and how it adversely affects the peripheral vascular system and promotes tissue injury.